PROJECT DESCRIPTION Pseudomonas aeruginosa ranks second among the most common human pathogens isolated from surgical sites, chronic infections, and burn wounds. Once established, growth of P. aeruginosa in biofilms makes it very difficult to eradicate the organisms by antimicrobial treatment. In order to eradicate P. aeruginosa biofilm infections, efforts should be focused on the developmental process leading to the formation of persistent and inherently resistant biofilms. We have evidence that autogenously produced pyruvate contributes to the formation and stability of established biofilms by P. aeruginosa. In turn, enzymatic depletion of pyruvate impaired biofilm formation and correlated with the disaggregation of established P. aeruginosa biofilms, with thus treated biofilm cells being rendered more susceptible to the aminoglycoside tobramycin and phagocytic monocytes. Preliminary data furthermore suggest enzymatic depletion of pyruvate to reduce the bacterial load in wounds. The goal of the proposed research project is to develop a nanodevice-based anti-biofilm therapy, based on pyruvate depletion, to reduce the biofilm burden, and enhance the efficacy of conventional antimicrobial treatment strategies of biofilm-related infections. The project is founded on the hypothesis that pyruvate depletion can be used as an anti-biofilm strategy to reduce the bacterial burden and enhance wound healing. As pyruvate depletion is accomplished using pyruvate dehydrogenase (PDH), which is unstable under in vivo conditions, we hypothesize that encapsulation of PDH into biodegradable nanoparticles will enhance/prolong PDH activity and stability, with in vitro selected optimal formulations being more efficient than free PDH in reducing the bacterial burden while enhancing killing of biofilm cells and wound healing in vivo. Experimentally, we will first assess in Aim 1 the applicability of pyruvate management in reducing the bacterial burden and enhancing wound healing in a porcine burn wound model and will determine the optimal PDH concentration required to so do. In Aim 2, we will develop biodegradable, biocompatible nanoparticles capable of encapsulating, maintaining and stabilizing PDH activity at concentrations required for the highest possible impact on biofilms in vivo while requiring the least amount of particles. Aim 3 is designed to select formulations that require the least number of nanoparticles to obtain the optimal PDH concentrations (high particle loading) while demonstrating comparable biofilm control capabilities at PDH activity levels that are equal to or lower than the optimal PDH. The two most promising nanoparticle formulations will be subsequently tested in Aim 4 for their efficacy in reducing the bacterial burden, enhancing the susceptibility of conventional antimicrobial agents in killing biofilm cells, and promoting wound healing in a porcine burn wound model. This proposed research brings together Drs. Karin Sauer (biofilm expert), Amber Doiron (expert in the fabrication of nanodevices), and Stephen Davis (expert on burn wounds) and is anticipated to result in innovative and effective therapeutic strategies, based on pyruvate depletion, to prevent, control and treat biofilm infections.